POS0495 CYTOKINE SYNERGY ENHANCES SYNOVIAL FIBROBLAST ACTIVATION IN CHILDREN WITH DOWN’S SYNDROME-ASSOCIATED ARTHRITIS
Authors: S. Foo, M. Canavan, V. Marzaioli, D. Veale, S. Wade, E. Macdermott, D. Deely, C. Foley, O. Killeen, U. Fearon
Journal: Annals of the Rheumatic Diseases (2022)

Background We have previously shown that children with Down’s syndrome-associated arthritis (DA) display a more aggressive form of inflammatory arthritis compared to that of Juvenile Idiopathic Arthritis (Shih et al. , 2019). DA is associated with an increase in polyfunctional T-cells coexpressing TNF-α DA is associated with an increase in polyfunctional T-cells coexpressing TNF-fibroblasts (FLS) (Foley et al. , 2019). Objectives In this study we examine the effect of cytokine synergy on primary DA FLS function. Methods Primary DA FLS were cultured and stimulated with TNF-α (0.1 and 1ng/ml), IL-17A (20 and 50ng/ml), IFN-ɣ (10 and 50ng/ml) and GM-CSF (20 and 100ng/ml) or a combination of these cytokines and the following functional experiments performed. Chemokine and adhesion molecule cell surface expression were quantified by flow cytometry, in addition to quantification of leukocyte-DA-FLS adhesion assays. Gene and protein expression of proinflammatory and metabolic mediators were quantified by ELISA and RT-PCR. Furthermore, real-time metabolic activity in response to cytokine stimulation was assessed by measuring the two major energy pathways: glycolysis (ECAR) and oxidative phosphorylation (OCR), by the Seahorse XFe96 Analyser. Results We examined the effects of T cell derived cytokines, TNF-α, IL-17A, IFN-γ and GM-CSF, alone and in combination on DA FLS function. TNF-α, IL-17A and IFN-γ induced IL-6, RANTES and MCP-1 production, with no effect observed for GM-CSF. Furthermore, TNF-α, IFN-ɣ and IL-17A increased leukocyte adhesion to DA FLS. TNF-α and IFN-ɣ induced cell surface expression of CXCR3, CXCR4, ICAM-1 and VCAM-1 on DA FLS. Next, we investigated the potential synergistic relationship that these cytokines could have on proinflammatory mediators. IL-17A and IFN-ɣ potentiated the effects TNF-α on IL-6 and MCP-1 secretion compared to stimulation alone. Furthermore, cytokine synergy significantly induced IL-6, IL-8, RANTES and LDHA mRNA expression compared to basal. IL-17A and IFN-αL-17A and IFN- eeased ts TNF-α on IL-6 and MCP-1 secretion compared to stimulation alone. Additionally, IFN the ECAR:OCR ratio demonstrating a shift in the metabolic profile of DA FLS to glycolysis. Overall DA FLS are transformed from a quiescent metabolic state to an energetic phenotype. Conclusion TNF-αNF-lusion:OCR ratio demonstrating a shift in the metabolhe aggressive phenotype of DA FLS through increased cytokine, adhesion molecule and chemokine expression, which is pathways for the treatment of DA. References [1]Foley, C. et al. (2019) ‘Increased T cell plasticity with dysregulation of T follicular helper, T peripheral helper and T regulatory cell responses in children with JIA and Down syndrome-associated arthritis’, Arthritis &amp; Rheumatology , pp. 0–1. doi: 10.1002/art.41150. [2]Shih, Y. J. et al. (2019) ‘Enthesitis-related arthritis is the most common category of juvenile idiopathic arthritis in Taiwan and presents persistent active disease’, Pediatric Rheumatology . Pediatric Rheumatology. doi: 10.1186/s12969-019-0363-0. Disclosure of Interests None declared.